BR112018013523B1 - HYDROGEL PRODUCT COMPRISING CROSS-LINKED GLYCOSAMINOGLYCAN MOLECULES AND PROCESS FOR PREPARING A HYDROGEL PRODUCT - Google Patents

Abstract

carbohydrate crosslinking agent. the invention relates to a hydrogel product comprising glycosaminoglycan molecules as the swellable polymer, wherein the glycosaminoglycan molecules are covalently cross-linked via cross-links consisting essentially of a spacer group selected from the group consisting of di-, tri-, tetra - and oligosaccharides.

Description

Technical Field of Invention

[001] The present invention relates to the field of hydrogels containing cross-linked polysaccharides and the use of such hydrogels in medical and/or cosmetic applications. More specifically, the present invention relates to hydrogels obtained from cross-linked glycosaminoglycans, particularly cross-linked hyaluronic acid, chondroitin or chondroitin sulfate.

Background of the Invention

[002] Water absorption gels, or hydrogels, are widely used in the biomedical field. They are usually prepared by chemical crosslinking of polymers to infinite networks. While many polysaccharides absorb water until completely dissolved, cross-linked gels of the same polysaccharides can typically absorb a certain amount of water until they are saturated, i.e., have a finite liquid holding capacity or degree of swelling.

[003] Hyaluronic acid, chondroitin and chondroitin sulfate are well-known biocompatible polymers. They are naturally occurring polysaccharides belonging to the group of glycosaminoglycans (GAGs). All GAGs are negatively charged heteropolysaccharide chains that have the ability to absorb large amounts of water.

[004] Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic use. HA is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example during visco surgery and as a dermal filler.

[005] Chondroitin sulfate (CS) is a highly abundant GAG found in mammalian connective tissues, where, along with other sulfated GAGs, it is bound to proteins as part of proteoglycans. It has previously been shown that CS-containing hydrogels can be successfully used in biomedical applications because of their similarity to the natural extracellular matrix ( Lauder, R.M., Complementary Ther Med. 17: 56-62, 2009 ). Chondroitin sulfate is also used in the treatment of osteoarthritis, for example as a dietary supplement.

[006] The cross-linking of glycosaminoglycans prolongs the life of the degradable polymers that make up the network, which is useful in many applications. However, cross-linking can also reduce the native properties of glycosaminoglycans. Therefore, it is typically desired to maintain a low degree of modification by efficient cross-linking to preserve the native properties and effects of the glycosaminoglycan itself.

Summary of the Invention

[007] It is an object of the present invention to provide a hydrogel having a glycosaminoglycan (GAG) as a wettable polymer.

[008] It is another object of the present invention to provide a method for crosslinking GAG molecules with reduced effect of the native properties of the GAG molecules.

[009] It is also an object of the present invention to provide a method for preparing hydrogels of GAG molecules in moderate and efficient ways.

[0010] For these and other purposes which will become apparent from this description, the present invention provides in accordance with a first aspect a hydrogel product comprising glycosaminoglycan molecules as the swellable polymer, wherein the glycosaminoglycan molecules are covalently cross-linked via cross-links comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides.

[0011] With reference to the inventive processes for preparing hydrogel products described herein, the term "crosslinker" refers to a molecule with two or more functional groups, particularly nucleophilic functional groups attached to a non-reactive spacer group, particularly a di-, tri-tetra- or oligosaccharide group.

[0012] Each of the two or more functional groups is capable of reacting with carboxylic acid groups on the GAG molecules to form stable covalent bonds. Preferably, the crosslinker consists of the two or more functional groups and the spacer.

[0013] With reference to the hydrogel products of the invention described herein, the term "cross-linking" refers to the portion, or residue, of the cross-linker by which the GAG molecules are covalently linked after cross-linking. Crosslinking typically consists of i) the spacer group and ii) the linking groups formed by reaction of the crosslinker functional groups with the carboxylic acid groups on the GAG. The spacer group may, for example, consist of a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose.

[0014] Crosslinking via crosslinking agents comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides provides a hydrogel product based entirely on carbohydrate-like structures or derivatives thereof, which minimizes the disturbance of cross-linking in the native properties of glycosaminoglycans. The di-, tri-, tetra- or oligosaccharide is preferably well defined in terms of structure and molecular weight. Preferably, the spacer is selected from a specific di-, tri-, tetra- or oligosaccharide structure. Preferably, the di-, tri-, tetra- or oligosaccharide is monodispersed or has a narrow molecular weight distribution. Using well-defined crosslinking agents based on di, tri, tetra or oligosaccharide together with a highly efficient condensation reaction allows the product to be assembled in a controlled manner. The crosslinker itself can also contribute to maintaining or enhancing the properties of the hydrogel, for example when crosslinking with a structure that correlates with hyaluronic acid (e.g. diamino hyaluronic acid tetrasaccharide) or when crosslinking with a structure with high retention properties. of water (eg trehalose).

[0015] The GAG can be, for example, sulfated or non-sulfated glycosaminoglycan, such as hyaluronan, chondroitin sulfate, heparan sulfate, heparosan, heparin, dermatan sulfate and keratan sulfate. In some embodiments, the GAG is hyaluronic acid, chondroitin, or chondroitin sulfate. In a preferred embodiment, the GAG is hyaluronic acid.

[0016] In preferred embodiments, the GAG is a native GAG. The GAG used in connection with the invention is preferably a natural GAG. The GAG is preferably used in its native state, i.e. the chemical structure of the GAG has preferably not been altered or modified by the addition of functional groups or the like. The use of GAG in its native state is preferred because this will provide a crosslinked structure closer to natural molecules, which retains the native properties and effects of GAG itself, and may minimize the immune response when crosslinked GAG is introduced into the body.

[0017] Covalently cross-linked GAG molecules consist of, or preferably consist essentially of, carbohydrate-like structures or derivatives thereof. This means that the cross-linked GAG molecules are preferably free, or essentially free of synthetic non-carbohydrate structures or linkers. This can be accomplished by using a GAG in its native state together with an inhibitor consisting of, or consisting essentially of, carbohydrate-like structures or derivatives thereof. The functional groups of the cross-linker are then covalently and directly linked to the carboxyl groups of the GAG. The crosslinks of the covalently crosslinked GAGs preferably consist of or consist essentially of di-, tri-, tetra- and oligosaccharide spacer groups.

[0018] The present invention provides, according to a second aspect, a process for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, comprising the steps of: (a) providing a solution of glycosaminoglycan molecules; (b) activating the carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a di- or multinucleic functional cross-linker comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides to obtain cross-linked glycosaminoglycan molecules.

[0019] The present invention involves the crosslinking of glycosaminoglycan molecules by covalent bonds, preferably amide bonds, typically using an activating agent for the carboxyl groups on the backbone of the glycosaminoglycan molecule and a di- or multinucleophile functional cross-linker comprising a selected spacer group from the group consisting of tri-, tetra- and oligosaccharides. Crosslinking according to the inventive method can be achieved in moderate and efficient ways, resulting in high yields with minimal degradation of the GAG molecules.

[0020] The functional di- or multinucleophile crosslinker contains a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides, which remains in the crosslinks between the GAG molecules. The di-, tri-, tetra- and functional di- or multinucleophilic oligosaccharides comprise at least two nucleophilic functional groups attached thereto. The at least two nucleophilic functional groups are preferably separated by the spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides.

[0021] The functional di- or multinucleophile crosslinker comprises two or more functional groups capable of reacting with functional carboxyl groups of the GAG, resulting in the formation of covalent bonds, preferably amide bonds. Nucleophilic functional groups are preferably capable of reacting with carboxyl groups on the glycosaminoglycan molecule to form amide bonds. In some embodiments, the nucleophilic functional groups of the di-, tri-, tetra- and oligosaccharides are selected from the group consisting of the primary amine, hydrazine, hydrazide, carbazate, semicarbazide, thiosemicarbazide, thiocarbazate and aminooxy.

[0022] The di-, tri-, tetra- and di- or multinucleophilic functional oligosaccharides can be derived from nucleophilic functional polysaccharides, such as chitobiose derived from chitin. The di-, tri-, tetra- and di- or multinucleophilic functional oligosaccharides can also be di-, tri-, tetra- and oligosaccharides that have been modified by the introduction of two or more nucleophilic functional groups.

[0023] A preferred group of crosslinkers with di- or multinucleophile functionality include homo- or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates and aminooxy.

[0024] In certain embodiments, the activation step (b) and the crosslinking step (c) occur simultaneously. In other embodiments, the activation step (b) occurs before and separately from the crosslinking step (c).

[0025] In a preferred embodiment, step (c) further comprises providing particles of the cross-linked GAG molecule having an average size in the range of 0.01-5 mm, preferably 0.1-0.8 mm .

[0026] In a preferred embodiment, the coupling agent of step (b) is a peptide coupling reagent. The peptide coupling reagent may be selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma and COMU. A preferred peptide coupling reagent is a triazine-based coupling reagent, including the group consisting of 4-(4,6-dimethoxy-1,3,5-triazyl-2-yl)-4-methylmorpholino chloride (DMTMM ) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), preferably DMTMM. Another preferred peptide coupling reagent is a carbodiimide coupling reagent, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

[0027] In a related aspect, the present invention also provides for the use of the hydrogel product as a medicine, such as in the treatment of soft tissue disorders. A method of treating a patient suffering from a soft tissue disorder is provided by administering to the patient a therapeutically effective amount of the hydrogel product. Also provided is a method of providing corrective or aesthetic treatment to a patient by administering to the patient a therapeutically effective amount of the hydrogel product.

[0028] Other preferred aspects and embodiments of the present invention will be apparent from the following detailed description of the invention and the appended claims. Detailed List of Preferred Embodiments 1. A hydrogel product comprising glycosaminoglycan molecules as the swellable polymer, wherein the glycosaminoglycan molecules are covalently cross-linked via cross-links comprising a spacer group selected from the group consisting of di-, tri-, tetra - and oligosaccharides. 2. A hydrogel product according to embodiment 1, wherein the glycosaminoglycan molecules are selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. 3. A hydrogel product according to embodiment 2, wherein the glycosaminoglycan molecules are hyaluronic acid. 4. A hydrogel product according to any of the preceding embodiments, wherein at least 75% of the crosslinks comprise a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. 5. A hydrogel product according to embodiment 4, wherein at least 90% of the crosslinks comprise a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. 6. A hydrogel product according to embodiment 5, wherein at least 95% of the crosslinks comprise a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. 7. A hydrogel product according to any of the preceding embodiments, wherein the spacer group is a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose. 8. A hydrogel product according to embodiment 7, wherein the spacer group is a hyaluronic acid tetrasaccharide or a hyaluronic acid hexasaccharide residue. 9. Hydrogel product according to embodiment 7, wherein the spacer group is a residue of trehalose, lactose, maltose, sucrose, cellobiose or raffinose. 10. A hydrogel product according to any of the preceding embodiments, wherein the spacer group is selected from the group consisting of di-, tri- and tetrasaccharides. 11. A hydrogel product according to any of the foregoing embodiments, wherein the crosslinks are linked to the glycosaminoglycan molecules by amide bonds. 12. A hydrogel product according to any of the foregoing embodiments, wherein at least 75% of the bonds between glycosaminoglycan molecules and crosslinks are amide bonds. 13. A hydrogel product according to embodiment 12, wherein at least 90% of the bonds between glycosaminoglycan molecules and crosslinks are amide bonds. 14. A hydrogel product according to embodiment 13, wherein at least 95% of the bonds between glycosaminoglycan molecules and crosslinks are amide bonds. 15. A hydrogel product according to any of the foregoing embodiments, wherein less than 5% of the bonds between glycosaminoglycan molecules and crosslinks are ether bonds. 16. A hydrogel product according to embodiment 15, wherein less than 1% of the linkages between glycosaminoglycan molecules and crosslinks are ester linkages. 17. A hydrogel product according to any of the preceding embodiments, wherein the cross-linked glycosaminoglycan molecule is in the form of gel particles having an average size in the range of 0.01-5 mm, preferably 0.1 -0.8 mm. 18. A hydrogel product according to any of the foregoing embodiments, in the form of an injectable formulation. 19. A process for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, comprising the steps of: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a functional di- or multinucleophile cross-linker comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides to obtain cross-linked glycosaminoglycan molecules. 20. Process according to embodiment 19, wherein the crosslinking of step (c) provides amide bonds between glycosaminoglycan molecules and crosslinking agents. 21. A process according to any one of embodiments 19-20, wherein the activation step (b) and the crosslinking step (c) occur simultaneously. 22. A process according to any one of embodiments 19-21, wherein the coupling agent and cross-linker are added to the glycosaminoglycan simultaneously. 23. A process according to any one of embodiments 19-20, wherein the activation step (b) occurs before and separately from the crosslinking step (c). 24. A process according to any one of embodiments 19-23, wherein step (c) further comprises providing particles of the cross-linked glycosaminoglycan having an average size in the range of 0.01-5 mm, preferably 0 .1-0.8 mm. 25. A process according to any one of embodiments 19-24, wherein the glycosaminoglycan molecules are selected from the group consisting of hyaluronic acid, chondroitin sulfate and chondroitin and mixtures thereof. 26. A process according to any one of embodiments 19-25, wherein the glycosaminoglycan molecules are hyaluronic acid. 27. A process according to any one of embodiments 19-26, wherein the coupling agent of step (b) is a peptide coupling reagent. 28. Process according to embodiment 27, wherein the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxime and COMU. 29. Process according to embodiment 28, wherein the peptide coupling reagent is a triazine-based coupling reagent. 30. Process according to embodiment 29, wherein the triazine-based coupling reagent is selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) chloride -4-methylmorpholino (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT). 31. A process according to embodiment 30, wherein the triazine-based coupling reagent is DMTMM. 32. Process according to embodiment 28, wherein the peptide coupling reagent is a carbodiimide coupling reagent. 33. A process according to embodiment 32, wherein the carbodiimide coupling reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS). 34. A process according to any one of embodiments 19-33, wherein the spacer group is a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose. 35. A process according to any one of embodiments 19-33, wherein the spacer group is a hyaluronic acid tetrasaccharide or hyaluronic acid hexasaccharide residue. 36. A process according to any one of embodiments 19-33, wherein the spacer group is a residue of trehalose, lactose, maltose, sucrose, cellobiose, or raffinose. 37. A process according to any one of embodiments 19-36, wherein the spacer group is selected from the group consisting of di-, tri- and tetrasaccharides. 38. A process according to any one of embodiments 19-37, wherein the nucleophilic groups of the crosslinker are selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semicarbazide, thiosemicarbazide, thiocarbazate and aminooxy. 39. Process according to embodiment 38, wherein the di-, tri-, tetra- and oligosaccharide nucleophilic groups are primary amine. 40. A process according to embodiment 39, wherein the crosslinker is a functional dinucleophile crosslinker. 41. The process according to embodiment 40, wherein the crosslinker is selected from the group consisting of diamino hyaluronic tetrasaccharide, diamino hyaluronic hexasaccharide, diamino trehalose, diamino lactose, diamino maltose, diamino sucrose, chitobiose or diamino raffinose. 42. A process according to any one of embodiments 19-41, further comprising the step: (d) subjecting the cross-linked glycosaminoglycan molecules obtained in step (c) to alkaline treatment. 43. Product obtainable by the process according to any of the modalities 19-42. 44. A hydrogel product according to any one of embodiments 118 and 43 for use as a medicament. 45. A hydrogel product according to embodiment 44 for use in treating soft tissue disorders. 46. Use of a hydrogel product according to any one of embodiments 1-18 and 43 for the manufacture of a medicament for the treatment of soft tissue disorders. 47. Method of treating a patient suffering from a soft tissue disorder by administering to the patient a therapeutically effective amount of a hydrogel product according to any one of modalities 1-18 and 43. 48. Method of providing treatment corrective or aesthetic treatment to a patient by administering thereto a therapeutically effective amount of a hydrogel product according to any one of embodiments 1-18 and 43. 49. A method of cosmetic skin treatment, comprising administering to the skin a skin care product. hydrogel according to any one of embodiments 1-18 and 43.

Detailed Description of the Invention

[0029] The present invention provides advantageous processes for the preparation of hydrogels obtained from cross-linked glycosaminoglycan (GAG) molecules, the resulting hydrogel products and their uses. GAGs are negatively charged heteropolysaccharide chains that have the ability to absorb large amounts of water. In the hydrogel products according to the invention, the cross-linked GAG molecule is the swellable polymer which provides the gel properties. The preparation process described herein is gentle on GAG molecules, but provides efficient cross-linking.

[0030] Thus, the present invention provides hydrogels of GAG molecules by cross-linking in aqueous media using a di- or multinucleophile functional cross-linker capable of forming covalent bonds directly with carboxylic acid groups of GAG molecules by a reaction involving the use of a coupling agent .

[0031] The GAG according to the invention is preferably selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In a preferred embodiment, the GAG molecule is hyaluronic acid. Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic use. HA is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example during visco surgery and as a dermal filler.

[0032] Unless otherwise indicated, the term "hyaluronic acid" encompasses all variants and combinations of variants of hyaluronic acid, hyaluronate or hyaluronan, of various chain lengths and states of charge, as well as with various chemical modifications. That is, the term also encompasses the various hyaluronate salts of hyaluronic acid with various counterions, such as sodium hyaluronate. Hyaluronic acid can be obtained from various animal and non-animal sources. Non-animal sources include yeast and preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1 to 10 MDa, but other molecular weights are possible.

[0033] The term "chondroitin" refers to GAGs having a repeating disaccharide unit consisting of alternating parts of unsulfated D-glucuronic acid and N-acetyl-D-galactosamine. For the avoidance of doubt, the term "chondroitin" does not encompass any form of chondroitin sulfate.

[0034] The term "chondroitin sulfate" refers to GAGs having a repeating disaccharide unit consisting of alternating parts of D-glucuronic acid and N-acetyl-D-galactosamine. The sulfate moiety may be present in several different positions. Preferred chondroitin sulfate molecules are chondroitin sulfate-4 and chondroitin sulfate-6.

[0035] Chondroitin molecules can be obtained from various animal and non-animal sources. Non-animal sources include yeast and preferably bacteria. The molecular weight of a single chondroitin molecule is typically in the range of 1 to 500 kDa, but other molecular weights are possible.

[0036] Cross-linked GAG comprises cross-links between the chains of the GAG molecule, which creates a continuous network of GAG molecules that is held together by covalent cross-links.

[0037] The chains of GAG molecules are preferably cross-linked to each other via cross-linking agents comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. It is preferred that the cross-linking agents are linked to the glycosaminoglycan molecules by amide bonds.

[0038] The cross-linked GAG product is preferably biocompatible. This implies that no, or only a very mild, immune response occurs in the treated individual. That is, none or only very mild undesirable local or systemic effects occur in the treated individual.

[0039] The cross-linked product according to the invention is a gel or a hydrogel. That is, it can be thought of as a water-insoluble, but substantially diluted, crosslinked system of GAG molecules when subjected to a liquid, typically an aqueous liquid.

[0040] The gel contains mostly liquid by weight and may contain, for example, 90-99.9% water, but behaves like a solid due to a three-dimensional network of cross-linked GAG molecules within the liquid. Due to its significant liquid content, the gel is structurally flexible and similar to natural tissue, which makes it very useful as a scaffold in tissue engineering and tissue augmentation. It is also useful for the treatment of soft tissue disorders and for corrective or aesthetic treatment. It is preferably used as an injectable formulation.

[0041] Crosslinking of the GAG molecule can be achieved by activation with a coupling agent, followed by reaction with a crosslinker. The concentration of the GAG molecule and the extent of crosslinking affect the mechanical properties, eg the elastic modulus G' and the gel stability properties. Gels of cross-linked GAG molecules can be characterized in terms of "degree of modification". The degree of modification of the GAG molecule gels generally ranges from 0.01 to 15% molar. The degree of modification (mol %) describes the amount of cross-linking agent(s) that is bound to the GAG molecule, i.e. the molar amount of cross-linking agent(s) bound relative to the total molar amount of repeating disaccharide units. The degree of modification reflects the extent to which the GAG molecule has been chemically modified by the crosslinker. The reaction conditions for activation and cross-linking and suitable analytical techniques for determining the degree of modification are all well known to those skilled in the art, who can easily adjust for these and other relevant factors and provide suitable conditions for obtaining a desirable degree of modification and verifying the resulting product characteristics in relation to the degree of modification.

[0042] The hydrogel product may also comprise a portion of GAG molecules that are not cross-linked, i.e., not linked to the network of three-dimensional cross-linked GAG molecules. However, it is preferred that at least 50% by weight, preferably at least 60% by weight, more preferably at least 70% by weight, and most preferably at least 80% by weight, of the GAG molecules in a gel composition form part of the network of cross-linked GAG molecules.

[0043] The cross-linked GAG molecule is preferably present in the form of gel particles. The gel particles preferably have an average size in the range of 0.01-5 mm, preferably 0.1-0.8 mm, such as 0.2-0.5 mm or 0.5-0. 8 mm.

[0044] The hydrogel product may be present in an aqueous solution, but may also be present in dry or precipitated form, for example in ethanol. The hydrogel product is preferably injectable.

[0045] The hydrogel product can be prepared by a process comprising the steps of: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a functional di- or multinucleophile cross-linker comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides to obtain cross-linked glycosaminoglycan molecules.

[0046] The GAG according to the invention is preferably selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In a preferred embodiment, the GAG molecule is hyaluronic acid.

[0047] In the activation step (b), the carboxyl groups on the GAG molecules are activated with a coupling agent to form activated GAG molecules.

[0048] In a preferred embodiment, the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma and COMU.

[0049] The peptide coupling reagent is preferably a triazine-based coupling reagent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholino chloride (DMTMM ) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT). A preferred triazine-based peptide coupling reagent is DMTMM.

[0050] Other preferred peptide coupling reagents are carbodiimide coupling reagents, preferably N-(3-dimethylaninopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

[0051] In the crosslinking step (c), the crosslinking of activated GAG molecules occurs through their carboxyl groups using a crosslinker. The cross-linker is a di- or multinucleophilic functional cross-linker comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides. The cross-linker connects the GAG chains to each other through carboxyl groups on the GAG backbone. The spacer group may, for example, be a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose residue. By the term "residue" it is meant herein that the structure of the compound is similar, but not identical to the proprietary compounds hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose, respectively. The structure of the residue may differ from the structure of the parent compound in that it was provided with two or more nucleophilic functional groups and optionally covalently linked through said nucleophilic functional groups to the carboxyl groups in the GAG backbone.

[0052] The functional di- or multinucleophile cross-linker comprises two or more functional groups capable of reacting with functional carboxyl groups of the GAG, resulting in the formation of covalent bonds, preferably amide bonds.

Diamino trealose (6,6'-diamino-6,6'-didesóxi trealose);

Diamino sacarose (6,6'-diamino-6,6'-didesóxi sacarose);

Quitobiose (2,2'-diamino-2,2'-didesóxi celobiose);

Diamino lactose (6,6´-diamino-6,6´-didesóxi lactose);

Tetrassacarídeo de ácido hialurônico reduzido N-desacetilado" ou tetrassacarídeo de ácido hialurônico de diamino reduzido"; e

"diaminorafinose (6,6 '- diamino-6,6' - didesóxi rafinose).[0053] A preferred group of crosslinkers with di- or multinucleophile functionality include homo- or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates and aminooxy. Non-limiting examples of such heterobifunctional cross-linking agents useful in the present invention include:

Diamino trehalose (6,6'-diamino-6,6'-dideoxy trehalose);

Diamino sucrose (6,6'-diamino-6,6'-dideoxy sucrose);

Chitobiose (2,2'-diamino-2,2'-dideoxy cellobiose);

Diamino lactose (6,6'-diamino-6,6'-dideoxy lactose);

N-deacetylated reduced hyaluronic acid tetrasaccharide" or reduced diamino hyaluronic acid tetrasaccharide"; and

"diaminoraffinose (6,6' - diamino-6,6' - dideoxy raffinose).

[0054] Reaction schemes 1a-1h schematically illustrate examples of coupling by heterobifunctional primary amine (1a), aminooxy (1b), carbazate (1c), semicarbazide (1d), thiosemicarbazide (1e), thiocarbazate (1f), hydrazine ( 1g) and hydrazide (1h)

[0055] The functional di- or multinucleophile cross-linker contains a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides, which remains in the cross-links between the GAG molecules.

[0056] The process can be carried out in a one-pot approach in aqueous media, involving the covalent coupling of di- or multinucleophilic functional cross-linking agents directly to carboxylic acid groups inherent in native GAGs using a suitable coupling agent. In a preferred embodiment, the activation step (b) and the crosslinking step (c) occur simultaneously.

[0057] In another embodiment, the activation step (b) occurs before and separately from the crosslinking step (c).

[0058] The process for generating the cross-linked hydrogel typically involves the preparation of a mixture of a GAG molecule, such as hyaluronic acid together with a cross-linker, such as diamino trehalose, DATH, (0.001 - 10 molar equivalents of amine to acidic groups). carboxylic acid, or preferably 0.001 - 1 molar equivalent) and a coupling agent such as DMTMM (0.01 - 10 molar equivalents for carboxylic acid groups, or preferably 0.05 - 1 molar equivalent). Incubation of the mixture at 5 - 50°C, preferably 10 - 40°C or even more preferred 20 - 35°C, for 2 - 120 hours, preferably 4 - 48 hours, followed by alkali treatment, neutralization, precipitation, washing and drying under vacuum, produces a cross-linked polysaccharide as a solid. The precipitate was swelled in phosphate buffer containing NaCl to form a hydrogel, the hydrogel being preferably micronized to hydrogel particles in the size of 0.01 - 5 mm, preferably 0.1 - 1 mm.

[0059] A typical application of the resulting hydrogel product involves the preparation of injectable formulas for the treatment of soft tissue disorders, including but not limited to corrective and aesthetic treatments.

[0060] In a more specific embodiment, crosslinking of chondroitin sulfate with DATH can be achieved as follows:

[0061] Diaminotrehalose (DATH) is synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the main Chain: Preparation and Characteristic Properties"; Keisuke Kurita, Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

[0062] Chondroitin Sulfate (CS) (10 - 200 kDa) is weighed in a Falcon tube. A master diaminetrehalose (DATH) solution is prepared by dissolving DATH in pH 7.4 phosphate buffer. The DMTMM is weighed into a container and the DATH solution is added to the DMTMM. The pH of the DMTMM-DATH solution is adjusted to approximately 7 by adding 0.2M HCl or 0.25M NaOH, and the mixture is subsequently added to CS. The contents are completely homogenized and then incubated at 15-55°C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh twice and swollen in NaOH. The gel is neutralized with 1.2 M HCl at pH 7 and ethanol precipitated. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The solid obtained is dried at 25°C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. Cross-linked CS gel is filled into syringes and sterilized.

[0063] In another more specific embodiment, the crosslinking of HA with diaminosucrose can be achieved as follows:

[0064] Diaminosucrose is prepared as described in "Library of mild and economic protocols for the selective derivatization of sucrose under microwave irradiation"; M. Teresa Barros, Krasimira T. Petrova, Paula Correia-da-Silva and Taterao M. Potewar; Green Chem., 2011, 13, 1897-1906.

[0065] Hyaluronic acid (HA) (10 - 1,000 kDa) is weighed into a container. A diaminosucrose master solution is prepared by dissolving diaminosucrose in pH 7.4 phosphate buffer. The DMTMM is weighed into a container and the diaminosucrose solution is added to the DMTMM. The pH of the diaminosucrose-DMTMM solution is adjusted to approximately 7 by addition of 0.2M HCl or 0.25M NaOH and the mixture is subsequently added to HA. The contents are completely homogenized and then incubated at 15-55°C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh twice and swollen in NaOH. The gel is neutralized with 1.2 M HCl at pH 7 and ethanol precipitated. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The solid obtained is dried at 25°C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The cross-linked HA gel is swollen in syringes and sterilized.

[0066] In another more specific embodiment, the crosslinking of HA with chitobiosis can be obtained as follows:

[0067] Hyaluronic acid (HA) (10 - 1000 kDa) is weighed into a container. A master chitobiose solution (purchased from Carbosynth Ltd. UK) is prepared by dissolving chitobiose in pH 7.4 phosphate buffer. The DMTMM is weighed into a container and the chitobiose solution is added to the DMTMM. The pH of the DMTMM-chitobiose solution is adjusted to approximately 7 by addition of 0.2M HCl or 0.25M NaOH and the mixture is subsequently added to HA. The contents are completely homogenized and then incubated at 15-55°C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh twice and swollen in NaOH. The gel is neutralized with 1.2 M HCl at pH 7 and then ethanol precipitated. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The solid obtained is dried at 25°C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The cross-linked HA gel is filled into syringes and sterilized.

[0068] In another more specific embodiment, crosslinking of HA with a reduced HA diaminotetrasaccharide can be achieved as follows:

[0069] Hyaluronic acid (HA) (10 - 1000 kDa) is weighed into a container. A master solution of a reduced diamino-HA tetrasaccharide is prepared by dissolving the reduced diamino-HA tetrasaccharide in pH 7.4 phosphate buffer. The DMTMM is weighed into a container and the reduced diamino-HA-tetrasaccharide solution is added to the DMTMM. The pH of the DMTMM and the reduction of the tetrasaccharidediamine-HA solution is adjusted to approximately 7 by the addition of 0.2M HCl or 0.25M NaOH and the mixture is subsequently added to HA. The contents are completely homogenized and incubated at 15-55°C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh twice and swollen in NaOH. The gel is neutralized with 1.2 M HCl at pH 7 and ethanol precipitated. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The solid obtained is dried at 25°C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and pressed through a filter mesh three times. The cross-linked HA gel is filled into syringes and sterilized.

[0070] In another more specific embodiment, crosslinking of HA with trehalose dicarbazate can be achieved as follows:

[0071] α,α -D-Trehalose (1 equiv.) (Anhydrous) (Carbosynth Ltd. UK) is dissolved in dry dimethylformamide (DMF), and triethylamine (2-6 equiv.) is added subsequently. The flask is cooled to 0°C (ice/water) and under a N2 atmosphere. 4-Nitrophenyl chloroformate (2-6 equiv.) is added to the vial dropwise. The resulting mixture is allowed to stir at room temperature for 2-48 h and then concentrated, purified by FC and dried in vacuo. The product is dissolved in DMF, and hydrazine monohydrate (2-20 equiv.) is added to the solution and stirred at 0-50°C for 4 - 48 h. The reaction is then concentrated, purified by FC and dried under vacuum to obtain, α,α-D-6,6'-dideoxy-6,6'-dicarbazate trehalose (dicarbazate trehalose, DCT).

[0072] Hyaluronic acid (HA) (10 - 1,000 kDa) is weighed into a container. A master solution of trehalose dicarbazate (DCT) is prepared by dissolving DCT in pH 7.4 phosphate buffer. The DMTMM is weighed into a container and the DCT solution is added to the DMTMM. The pH of the DMTMM-DCT solution is adjusted to approximately 7 by addition of 0.2M HCl or 0.25M NaOH and the mixture is subsequently added to HA. The contents are completely homogenized and incubated at 15-55°C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh twice and swollen in NaOH. The gel is neutralized with 1.2 M HCl at pH 7 and ethanol precipitated. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The solid obtained is dried at 25°C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The cross-linked HA gel is filled into syringes and sterilized.

[0073] In another more specific modality, the crosslinking of HA with diaminoxytrehalose can be obtained as follows:

[0074] To a stirred suspension of α,α-D-trehalose (1 equiv.) (anhydrous) (Carbosynth Ltd. UK) in anhydrous THF, N-hydroxyphthalimide (2-10 equiv.) and triphenylphosphine (2-10 equiv. .) is added, and the mixture is stirred for 5-60 min. The diisopropyl azodicarboxylate (DIAD, 2-10 equiv.) is then added dropwise at 0 - 40°C and the mixture is stirred for 2-48 h at 0 - 40°C. The solvent is removed under vacuum and the crude product is purified by FC and dried under vacuum. A suspension of the product in a mixture of MeOH and CH2Cl2 treated with hydrazine monohydrate (2-20 equiv.) and the mixture stirred at 0-40°C for 2-24 h, followed by concentration, purification by FC and vacuum drying to obtain diaminooxytetralose.

[0075] Hyaluronic acid (HA) (10 - 1000 kDa) is weighed into a container. A diamineoxytrehalose (DAOT) master solution is prepared by dissolving DAOT in pH 7.4 phosphate buffer. The DMTMM is weighed into a container and the DAOT solution is added to the DMTMM. The pH of the DMTMM-DAOT solution is adjusted to approximately 7 by adding 0.2M HCl or 0.25M NaOH and the mixture is subsequently added to HA. The contents are completely homogenized and incubated at 15-55°C for 2-48 h. The resulting material is pressed through a 1 mm steel mesh twice and swollen in NaOH. The gel is neutralized with 1.2 M HCl at pH 7 and ethanol precipitated. The resulting precipitate is washed with 100 mM NaCl in 70% ethanol, with 70% ethanol and ethanol. The solid obtained is dried at 25°C under vacuum. The precipitate is swelled in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The cross-linked HA gel is filled into syringes and sterilized.

EXAMPLES

[0076] Without wishing to be limited thereto, the present invention will be illustrated below by way of examples.

Definitions and Analysis

[0077] SwF - Swell factor analysis was performed in brine.

[0078] [PS] - Polysaccharide concentration, eg HA concentration. PS concentration was measured with LC-SEC-UV or NIR.

[0079] GelP - Part of Gel (also known as gel content or GeIC) is a description of the percentage of polysaccharide that is part of the gel network. A number of 90% means that only 10% of the polysaccharide is not part of the network. The amount of free polysaccharide in the gel was measured with LC-SEC-UV.

[0080] SwC - swelling capacity is the total liquid absorption of one gram of polysaccharide, not corrected for the gel part.

[0081] SwCC - Corrected swelling capacity (also sometimes referred to as SwDC) is the total liquid uptake of one gram of polysaccharide, corrected for the gel portion.

[0082] CrR - Effective cross-linking ratio was analyzed with LC-SEC-MS and defined as: CrR = mol of cross-linked cross-linker with amide bonds mol of cross-linked cross-linker with amide bonds

[0083] A CrR of 1.0 means that the entire crosslinker is crosslinked. Alkaline or thermal hydrolysis

[0084] In some of the examples below, the product was subjected to alkaline hydrolysis or heat in order to hydrolyze ester bonds formed during the crosslinking process. Alkaline/thermal hydrolysis results in only cross-linking amide bonds in the final product. Alkaline/thermal hydrolysis was performed as follows:

alkaline hydrolysis

[0085] The material was swelled in 0.25 M NaOH (1 g of material: 9 g of 0.25 M NaOH resulting in pH 13) for at least 1 hour at room temperature. The gel was neutralized with 1.2M HCl at pH 7 and then ethanol precipitated. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagents, then with 70% ethanol to remove salts, and finally with ethanol to remove water. Ethanol was removed in a vacuum dryer overnight.

[0086] The precipitate was swollen in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a fine mesh filter three times. The gel was filled into syringes and sterilized. In some cases, some of the syringes were not sterilized to see the effect of sterilization.

thermal hydrolysis

[0087] The material was swelled in 0.7% NaCl phosphate buffer pH 7.4 at room temperature. The pH was adjusted to 7.2-7.5 if necessary. The gel was left at 70°C for 20-24 h and then reduced in particle size through a fine filter mesh three times. The gel was filled into syringes and sterilized. In some cases, some of the syringes were not sterilized to see the effect of sterilization.

Hyaluronic diaminotrehalose synthesis

[0088] Diaminotrehalose (DATH) was synthesized as described in "Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties" Keisuke Kurita * Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

Example 1 - Crosslinking of hyaluronic acid with diaminotrehalose (DATH)

[0089] Several experiments were performed (Examples 1-1 to 1-5) that involved cross-linking hyaluronic acid (HA) of different molecular weights with various molar ratios of DATH using 4-(4,6-dimethoxy-1, 3,5-triazin-2-yl)-4-methylmorpholino (DMTMM) as a coupling agent. The relationships between HA, DATH and DMTMM are shown in Table 1 below.

[0090] Hyaluronan (molecular weight (Mw) from about 100 kDa to about 1000 kDa) was weighed in a Falcon tube. A master diaminetrehalose (DATH) solution was prepared by dissolving DATH (0.001 - 0.005 equiv.) in phosphate buffer pH 7.4. DMTMM (0.05 equiv.) was weighed into a PTFE container and the DATH solution was added to DMTMM to dissolve it. The DMTMM-DATH solution was adjusted to pH 6-7 with 1.2M HCl or 0.25M NaOH and then added to the HA. The contents were completely homogenized and then incubated at 35°C for 24 h.

[0091] The resulting material was pressed through a 1 mm steel mesh twice and then treated with a NaOH solution. The gel was neutralized with 1.2M HCl at pH 7 and then ethanol precipitated. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagents, then with 70% ethanol to remove salts, and finally with ethanol to remove water. Ethanol was removed in a vacuum dryer overnight.

NA - não disponível, pois a análise não foi feita[0092] The precipitate was swollen in 0.7% NaCl phosphate buffer pH 7.4 and then pressed through a filter mesh three times. The gel was filled into syringes and sterilized.

NA - not available as the analysis was not performed

Example 2 - Cross-linking of hyaluronic acid with diaminotrehalose (DATH)

[0093] Several experiments were performed (Examples 2-1 to 2-11) that involved cross-linking hyaluronic acid (HA) of different molecular weights with various molar ratios of DATH using 4-(4,6-dimethoxy-1, chloride). 3,6-triazin-2-yl)-4-methylmorpholino (DMTMM) as a coupling agent. The proportions between HA, DATH and DMTMM are shown in Table 2 below.

Células vazias - nenhuma análise feita.[0094] Hyaluronic acid was weighed into a reaction vessel. A crosslinker master solution (DATH) was prepared by dissolving it in pH 7.4 phosphate buffer. The DMTMM was weighed into a PTFE container and the crosslinker solution was added to the DMTMM to dissolve it. The pH of the DMTMM crosslinker solution was adjusted to 6-7 with 1.2M HCl or 0.25M NaOH and then added to the HA. The contents were completely homogenized and then incubated at 35°C for 24 h. The resulting material was pressed through a 1 mm steel mesh twice and then treated with heat or alkali. The results are shown in Table 2. Table 2. Hyaluronic acid crosslinking summary with (DATH)

Empty cells - no analysis done.

Example 3 - Crosslinking of Hyaluronic Acid with Chitobiose (CB)

[0095] Hyaluronic acid was weighed into a reaction vessel. A crosslinker master solution (chitobiosis) was prepared by dissolving it in pH 7.4 phosphate buffer. The DMTMM was weighed into a PTFE container and the crosslinker solution was added to the DMTMM to dissolve it. The DMTMM crosslinker solution was adjusted to pH 6-7 with 1.2M HCl and then added to the HA. The contents were completely homogenized and then incubated at 35°C for 24 h. The resulting material was pressed through a 1 mm steel mesh twice and then heat or alkali treated according to general procedures. The proportions between HA, chitobiose and DMTMM are shown below in Table 3 (Examples 3-1 to 3-2).

Example 4 - Cross-linking of hyaluronic acid with diaminotetra-HA (DA-4HA)

[0096] Diaminotetra-HA (DA-4HA) was synthesized according to the scheme below:

Step 1

[0097] A solution of HA-4 (500 mg, 0.61 mmol) in water (5 mL) at room temperature was treated with sodium borohydride (23.05 mg, 0.61 mmol) and the resulting solution was stirred for 3 h, concentrated to dryness to provide reduced product 1 (532 mg, assuming 100%) as a white foam. LCMS (tr = 0.28 min., ES + = 779.4 (M-2 Na + 2H)

step 2

[0098] Reduced product 1 (532 mg) was dissolved in aqueous NH 2 OH (5 mL, 50% v/v/) and solid NH 4 I (100 mg) was added. The resulting suspension was heated at 70 °C for 48 h, cooled to room temperature and concentrated to dryness to provide a residue. The residue was precipitated in pure EtOH and the resulting precipitate was collected by filtration and dried to a constant weight to provide the 1:1 mixture of diamine 2 and monoamine 3 in quantitative yield. The crude reaction product was used without further purification. 2: LCMS (tr = 0.16 min., ES + = 695.36 (M-2 Na + 2H) 3: LCMS (tr = 0.19 min., ES + = 737.47 (M-2 Na + 2H)

[0099] Hyaluronic acid was weighed into a reaction vessel. A crosslinker master solution (diaminotetra-HA), synthesized as described above, and DMTMM respectively were prepared by dissolving it in pH 7.4 phosphate buffer. The pH of the solutions was adjusted to 7 and then added to the HA. The contents were completely homogenized and then incubated at 23°C for 24 hours. The resulting material was pressed through a 1 mm steel mesh twice and then heat treated according to general procedures. The relationship between HA, diaminotetra-HA and DMTMM is shown below in Table 3 (Example 4).

Example 5 - Cross-linking of Heparosan (HEP) with diaminotrehalose (DATH)

[00100] The DMTMM coupling agent and the DATH crosslinker were weighed into separate reaction vessels and dissolved in phosphate buffer (pH 7.4). The pH of the solutions was adjusted to pH 7-7.5 with 1.2 M HCl or 0.25 M NaOH. Subsequently, the DMTMM and DATH solutions were successively added to the heparosan weighed in a reaction vessel. The contents were completely homogenized and then incubated at 35°C for 24 h. The resulting material was pressed through a 1 mm steel mesh twice and then heat treated according to general procedures. The ratios of heparosan, DATH and DMTMM are shown in Table 3 below (Examples 5-1 to 5-2).

Example 6 - Crosslinking of Chondroitin Sulfate (CS) with Diaminotrehalose (DATH)

PS = polissacarídeo, HA = hialuronano, HEP = heparosan, CS = sulfato de condroitina, DA-4HA = diaminotetra-HA, CB = quitobiose Células vazias - nenhuma análise realizada.[00101] The DMTMM coupling agent and the DATH crosslinker were weighed into separate reaction vessels and dissolved in phosphate buffer (pH 7.4). The pH of the solutions was adjusted to pH 7-7.5 with 1.2 M HCl or 0.25 M NaOH. Subsequently, the DMTMM and DATH solutions were successively added to the chondroitin sulfate weighed in a reaction vessel. The contents were completely homogenized and then incubated at 35°C for 24 h. The resulting material was pressed through a 1 mm steel mesh twice and then heat treated according to general procedures. The relationships between chondroitin sulfate, DATH and DMTMM are shown below in Table 3 (Examples 61 to 6-2). Table 3. Summary of Crosslinking Examples 3-6

PS = polysaccharide, HA = hyaluronan, HEP = heparosan, CS = chondroitin sulfate, DA-4HA = diaminotetra-HA, CB = chitobiose Empty cells - no analysis performed.

Claims (15)

1. Hydrogel product CHARACTERIZED in that it comprises glycosaminoglycan molecules as the swellable polymer, wherein the glycosaminoglycan molecules are covalently cross-linked via cross-links consisting essentially of a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides, wherein the cross-linked glycosaminoglycan molecules are free, or essentially free of synthetic structures or linkers other than carbohydrate. 2. Hydrogel product, according to claim 1, CHARACTERIZED by the fact that the glycosaminoglycan molecules are hyaluronic acid. 3. Hydrogel product, according to claim 1 or 2, CHARACTERIZED in that the spacer group is a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose. 4. Hydrogel product according to any one of claims 1 to 3, CHARACTERIZED in that the spacer group is selected from the group consisting of di-, tri- and tetrasaccharides. 5. Hydrogel product, according to any one of claims 1 to 4, CHARACTERIZED by the fact that at least 90% of the bonds between glycosaminoglycan molecules and crosslinks are amide bonds. 6. Hydrogel product, according to any one of claims 1 to 5, CHARACTERIZED by the fact that less than 5% of the bonds between glycosaminoglycan molecules and cross-links are ester bonds. 7. Hydrogel product, according to any one of claims 1 to 6, CHARACTERIZED in that it is in the form of an injectable formulation. 8. Hydrogel product according to any one of claims 1 to 7, CHARACTERIZED by the fact that the glycosaminoglycan is hyaluronic acid and the crosslinks consist essentially of trehalose. 9. Process for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, CHARACTERIZED in that it comprises the steps of: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; (c) cross-linking the activated glycosaminoglycan molecules through their activated carboxyl groups using a di- or multinucleophile functional cross-linker comprising a spacer group selected from the group consisting of di-, tri-, tetra- and oligosaccharides to obtain cross-linked glycosaminoglycan molecules, in that the cross-linked glycosaminoglycan molecules are free, or essentially free of synthetic structures or linkers other than carbohydrate. 10. Process, according to claim 9, CHARACTERIZED by the fact that the glycosaminoglycan molecules are hyaluronic acid. 11. Process according to claim 9 or 10, CHARACTERIZED by the fact that the spacer group is a residue of hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose. 12. Process according to any one of claims 9 to 11, CHARACTERIZED in that the spacer group is selected from the group consisting of di-, tri- and tetrasaccharides. 13. Process according to any one of claims 9 to 12, CHARACTERIZED by the fact that the nucleophilic groups of the crosslinker are selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semicarbazide, thiosemicarbazide, thiocarbazate and aminooxy. 14. Process according to any one of claims 9 to 13, CHARACTERIZED by the fact that the crosslinking of step (c) provides amide bonds between glycosaminoglycan molecules and crosslinkers. 15. Process according to any one of claims 9 to 14, CHARACTERIZED by the fact that the coupling agent is a triazine-based coupling reagent, preferably DMTMM.